Difluoroglutamic acid conjugates with folates and anti-folates for the treatment of neoplastic diseases

ABSTRACT

This invention relates to intermediates for the preparation of certain conjugates of folates and antifolates with difluoroglutamic acid which are useful in the treatment of patients suffering from certain neoplastic diseases including leukemia, melanomas, carcinomas, sarcomas and mixed neoplasias.

FIELD OF THE INVENTION

This invention relates to novel difluoroglutamic acid conjugates withfolates and anti-folates, to the use of these conjugates in thetreatment of neoplastic diseases, and to intermediates used in thepreparation of the conjugates.

BACKGROUND OF THE INVENTION

Folates and classical antifolates such as MTX¹ are convertedintracellularly to poly(γ-glutamyl) metabolites by the enzymefolylpolyglutamate synthetase.

    PteGlu.sub.n+ ATP+L-Glu→PteGlu.sub.n+1+ ADP+P.sub.i

Since it is now known that folylpolyglutamates are essential to theproper functioning of folate metabolism, and antifolylpolyglutamates areimplicated in the cytotoxic action of classical antifolates such as MTX,folylpolyglutamate synthetase has become an important enzyme for studyin folate biochemistry and biochemical pharmacology. In this regard, thespecificity of this enzyme for pteroyl and L-glutamate substrates hasbeen extensively investigated. For pteroyl substrates, the structure ofthe heterocyclic component can vary considerably, but a terminalL-glutamate residue has been shown to be absolutely required forsubstrate activity in all reports to this time. Specificity for theincoming amino acid is strict, but not absolute. L-homocysteic acid andD,L-ervthro- or D,L -threo-4-fluoroglutamate can all serve as efficientalternate substrates, but in each case incorporation causes chaintermination. Chain termination by the 4-fluoroglutamate diastereomersdemonstrates the stringent specificity for L-glutamate at the γ-g1utamylacceptor site.

F₂ Glu is a potent, concentration-dependent inhibitor ofpoly(γ-glutamylation) using [³ H]Glu and either methotrexate (4-NH₂-10-CH₃ PteGlu) or tetrahydrofolate as substrates. Applicants havedetermined that F₂ Glu acts as an alternate substrate, but in contrastto the previously characterized alternate substrate 4-fluoroglutamate(McGuire and Coward, J. Biol. Chem. 260: 6747 (1985)), it did notterminate polyglutamate chain elongation. Instead, F₂ Glu promotes chainelongation. Thus, synthesis of products from [³ H]methotrexatecontaining 1 and 2 additional amino acid residues occurs at asubstantially higher rate in the presence of F₂ Glu when compared toidentical reactions in the presence of Glu; this is more pronounced forthe product containing 2 additional residues. The increased rate ofaddition is not solely a function of ligating F₂ Glu to the internal Gluor to a previously incorporated F₂ Glu, since ligation of Glu to 4-NH₂-10-CH₃ PtGlu-γ-(3,3-difluoroglutamate) is also enhanced. These resultsare consistent with F₂ Glu enhancing the synthesis of poly(γ-g1utamate)metabolites at the level of either the incoming amino acid (glutamateanalog) or the γ-glutamyl acceptor species. F₂ Glu is thus the firstglutamate analog which enhances chain elongation catalyzed byfolylpolyglutamate synthetase.

SUMMARY OF THE INVENTION

The compound 3,3-difluoroglutamate (F₂ Glu) is used to prepareconjugates with certain folates and antifolates of formula 1 ##STR1##wherein R₁ is --NH₂, H, or --CH_(3;) R₂ is --NH₂ or --OH;

R₃ is H, (C₁₋ C₄)alkyl, allyl, or propargyl, and

X, Y, and Z are each independently a nitrogen atom or a CH group;

and the pharmaceutically acceptable salts thereof. The structure 1conjugates are useful in the treatment of tumors and psoriasis. Moreoverthe compound F₂ Glu and certain PABAF -F₂ Glu compounds areintermediates used in the preparation of the formula 1 compounds.

DETAILED DESCRIPTION OF THE INVENTION

The difluoro glutamate portion of the compounds of this inventionpossess an achiral center and thus the compounds of this invention existas pairs of stereochemical isomers. While the enantiomer, correspondingto the natural configuration of L-glutamic acid, are preferred,applicants contemplate that both the individual isomers and mixtures ofthe individual isomers including the raceamic mixture are within thescope of this invention. Unless otherwise indicated the compounds ofthis application are mixtures of the two stereoisomers. Further, thosecompounds of formula 1 wherein Z is a CH group and wherein R3 is otherthan a hydrogen have a second achiral site. The stereochemicalconfiguration around this second achiral site is not critical andapplicants again intend that the separate isomers and mixtures beincluded within the scope of this invention. A racemic mixture ofisomers with regard to this second achiral site is preferred.

The compounds of formula 1 contain two carboxylic acid moieties and canform mono or di basic salts. Illustratively, these salts include thoseof alkali metals, as for example, sodium and potassium; alkaline earthmetals, such as calcium and magnesium; light metals of Group IIIAincluding aluminum; and organic primary, secondary and tertiary amines,as for example, trialkylamines, including triethylamine, procaine,dibenzylamine, 1-ethenamine, N,N'-dibenzylethylenediamine,dihydroabietylamine, N-(lower)alkylpiperidine, and any other suitableamine. Sodium salts are preferred.

The compounds of formula 1 can also form pharmaceutically acid additionacceptable salts with any non-toxic, organic or inorganic acid.Illustrative inorganic acids which form suitable salts includehydrochloric, hydrobromic, sulphuric and phosphoric acid and acid metalsalts such as sodium monohydrogen orthophosphate and potassium hydrogensulfate. Illustrative organic acids which form suitable salts includethe mono, di and tricarboxylic acids. Illustrative of such acids are,for example, acetic, glycolic, lactic, pyruvic, malonic, succinic,glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic,hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic,salicylic, 2-phenoxybenzoic and sulfonic acids such as methane sulfonicacid and 2-hydroxyethane sulfonic acid. Hydrochloride salts arepreferred.

The compounds of formula 1 can be prepared by protecting group removalfrom the product of the reaction of the formula 2 folate or antifolatederivative ##STR2## wherein R₁, R₂, R₃, X, Y, and Z are as defined abovefor the formula 1 compounds and a suitably protected derivative of F₂Glu such as the t-butyl ester derivative of formula 3, F₂ Glu(OtBu)₂.##STR3## The coupling of the formula 2 compound and the protected F₂ Gluderivative of formula 3 can be accomplished is any appropriate mannerfor preparing an amide from an amine and a carboxylic acid. Applicantshave coupled the formula 2 and 3 compounds using diethylphosphorocyanidate ((EtO)₂ P(═O)CN). This reaction is performed byadding (slow dropwise addition) a solution of the appropriate formula 2compound to a solution of ((EtO)₂ P(═O)CN) and an acid scavenger such aspyridine, or preferably triethylamine. The resulting mixture is allowedto react at from about 0° C. to about 60° C., preferably at ambienttemperature for from about 1 hour to about 10 hours, preferably fromabout 2 to about 5 hours, until the formula 2 compound is substantiallyreacted with the phosphorocyanidate. To this mixture is then added asolution of F₂ Glu(OtBu)2 and the resulting reaction is allowed toproceed for from about 24 hours to about 100 hours, generally for about72 hours at from about 0° C. to about 60° C., preferably at ambienttemperature. The crude product is isolated, for example, by solventevaporation, washed with water and dried. The crude product is thentreated with trifluoroacetic acid (neat) to remove the t-butyl esterprotecting groups. The desired formula 1 compound is isolated andpurified, for example, by column chromatography. Suitable solvents forthe above described coupling reaction include any solvent in which thevarious reactants are soluble and which do not interfere with thereaction such as preferably dimethylformamide (DMF).

The compounds of formula 1 wherein Z is a nitrogen atom can be preparedby an alternative procedure in which a p-aminobenzoic acid (PABA)derivative of formula 4 ##STR4## wherein R₃ is as defined above for theformula 1 compounds is reacted with the formula 3 compound, F₂Glu(OtBu)₂, to give the intermediate compound of formula 5 ##STR5##wherein R₃ is as defined above for the formula 1 compounds. Thiscoupling can be accomplished in any suitable manner such as by theprocedures described in McGuire, et al., Cancer Research 1989, 49,4517-4525; Galican et al., Proc. Natl. Acad. Sci., USA, 1985, 82,2598-2602; Piper and Montgomery, J. Org. Chem., 42(2) 208-211 (1977);Taylor, et al., J. Med. Chem. 28, 913-921 (1985); Taylor, et al., J.Med. Chem. 28, 1517-1522 (1985); Jones, et al., J. Med Chem. 32, 847-852(1989).

The formula 5 intermediate is then condensed with the bromomethylderivative of formula 6 ##STR6## wherein R₁, R₂, X and Y are as definedfor the formula 1 compounds above to produce the t-butoxy protectedcompounds which upon hydrolysis to remove the t-butoxy protecting groupssuch as with trifluoroacetic acid yield the desired compounds offormula 1. This reaction can be accomplished by allowing a mixture ofthe formulae 5 and 6 compounds and dimethylacetamide (Me₂ NAc) to reactfor about 2 to about 12 hours, preferably for about 4 hours, at atemperature of from about 25° C. to about 80° C., preferably at about50-55° C., and then leaving the reaction mixture stand at ambienttemperature for from about 6 to about 24 hours, preferably for about 12hours. The Me₂ NAc is then removed under reduced pressure and the crudematerial placed in an ice bath and treated with trifluoroacetic acid,allowing the mixture to warm to room temperature after addition iscomplete. After standing for from about 1 to about 6 hours, typicallyfor about 4 hours, the TFA is removed under reduced pressure to give thecrude desired product of formula 1. This product can be purified by, forexample, chromatography (DEAE-cellulose with an NH₄ HCO₃ gradient). Thiscondensation reaction is more completely described in the referencesmentioned in the preceding paragraph.

The selection and utilization of particular blocking groups are wellknown to one of ordinary skill in the art. In general, blocking groupsshould be selected which adequately protect the amino or hydroxy groupsin question during subsequent synthetic steps and which are readilyremovable under conditions which will not cause degradation of thedesired product. Examples of suitable hydroxy protecting groups are C₁₋C₆ alkyl, tetrahydropyranyl, methoxymethyl, methoxyethoxy-methyl,t-butyl, benzoyl, and triphenylmethyl. The term C₁₋ C₆ alkyl refers to asaturated hydrocarbyl radical of one to six carbon atoms of straight,branched, or cyclic configuration. The benzoylated derivative can beformed by reacting the unblocked compound with benzoyl chloride in thepresence of pyridine. Examples of suitable amino protecting groups arebenzoyl, formyl, acetyl, trifluoroacetyl, phthalyl, tosyl,benzenesulfonyl, benzyloxycarbonyl, substitutedbenzyloxycarbonyl (e.g.,p-chloro,p-bromo, p-nitro, p-methoxy, o-chloro, 2,4-dichloro, and2,6-dichloro derivatives), t-butyloxycarbonyl (Boc), t-amyloxycarbonyl,isopropyloxycarbonyl, 2-(p-biphenyl)-isopropyloxycarbonyl,allyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl,adamantyloxycarbonyl, phenylthiocarbonyl, and triphenylmethyl. Preferredamino protected compounds include the benzoyl derivative, made byreacting the unblocked compound with benzoyl chloride, and the acetylderivative, made by reacting the unblocked compound with aceticanhydride.

The present invention provides a method of treating a patient sufferingfrom a neoplastic disease comprising administering to the patient atherapeutically effective antineoplastic amount of a compound offormula 1. By administering a therapeutically effective antineoplasticamount of a compound of formula 1 to a patient suffering from aneoplastic disease, an antineoplastic effect is provided. The term"patient" refers to a warm-blooded animal, such as primates, includinghumans, sheep, horses, cattle, pigs, dogs, cats, rats and mice.

The term "neoplastic disease" as used herein refers to an abnormal stateor condition characterized by rapidly proliferating cell growth orneoplasm. Based upon standard laboratory experimental techniques andprocedures well known and appreciated by those skilled in the art, aswell as upon comparisons with compounds of known usefulness, thecompounds of formula 1 are useful in the treatment of patients sufferingfrom those neoplastic diseases which generally are or can be treatedwith folates and antifolates such as methotrexate, aminopterin,5,10-dideazafolate and leucovorin. Such neoplastic diseases include:leukemias, including but not limited to acute lymphoblastic, chroniclymphocytic, acute myloblastic and chronic mylocytic; carcinomas,including but not limited to those of the cervix, esophagus, stomach,small intestines, colon and lungs; sarcomas, including but not limitedto oesteroma, osteosarcoma, lipoma, liposarcoma, hemangioma andhemangiosarcoma; melanomas, including amelanotic and melanotic; andmixed types of neoplasias such as, for example, carcinosarcoma, lymphoidtissue type, folicullar reticulum, cell sarcoma and Hodgkins Disease. Ofcourse, one skilled in the art will recognize that not every compound offormula 1 will be effective against each of the neoplastic diseasestates, and that selection of the most appropriate compound is withinthe ability of one of ordinary skill in the art and will depend on avariety of factors including assessment of results obtained in standardanimal tumor models. In general the compounds of formula 1 are useful inthe treatment of those neoplastic diseases currently treated withfolates and antifolates. The compounds of this invention are expected tobe more potent and more selective than are the non-conjugated folatesand antifolates. The added potency of the compounds of this invention isbelieved to result from the tendency of the compounds to promoteglutamyl chain enlogation and thereby cause accumulation of the toxicfolate and antifolates within the cell.

The term "antineoplastic effect" and the term "treating a neoplasticdisease" refers to an effect of controlling the growth or proliferationof the neoplasm or in prolonging the survivability of the patient beyondthat expected in the absence of such treatment. The growth orproliferation of a neoplasm is controlled by slowing, interrupting,arresting or stopping its growth, proliferation or its metastases. Theterm "treating a neoplastic disease" therefore does not necessarilyindicate a total elimination of the neoplastic disease. It is believedthat prolonging the survivability of a patient, beyond being asignificant advantageous effect in and of itself, also indicates thatthe growth of the neoplastic disease has been controlled.

In effecting treatment of a patient afflicted with a neoplastic diseasedescribed above, a compound of formula 1 can be administered in any formor mode which makes the compound bioavailable in therapeuticallyeffective antineoplastic amounts, including oral and parenteral routes.For example, compounds of formula 1 can be administered orally,topically, subcutaneously, intramuscularly, intravenously,transdermally, intranasally, rectally, and the like. Oral administrationis generally preferred. One skilled in the art can readily select theproper form and mode of administration depending upon the particularcharacteristics of the compound selected the disease state to betreated, the stage of the disease, and other relevant circumstances.

As used herein, the term "therapeutically effective antineoplasticamount" refers to an amount of the compound of formula 1 which iseffective in providing an antineoplastic effect. A therapeuticallyeffective antineoplastic amount can be readily determined by theattending diagnostician, as one skilled in the art, by the use of knowntechniques and by observing results obtained under analogouscircumstances. In determining the therapeutically effectiveantineoplastic amount, a number of factors are considered by theattending diagnostician, including, but not limited to: the species ofmammal; its size, age, and general health; the specific diseaseinvolved; the degree of or involvement or the severity of the disease;the response of the individual patient; the particular compoundadministered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A therapeutically effective antineoplastic amount of a compound offormula 1 is expected to vary from about 0.1 milligram per kilogram ofbody weight per day (mg/kg/day) to about 500 mg/kg/day. Preferredamounts are expected to vary from about 1 to about 20 mg/kg/day. Theseranges are particularly reflective of effective amounts upon oraladministration but also are reflective of the operable ranges forparenteral administration. Preferably from 1 to 4 daily doses would beadministered typically with from 5 mg to 100 mg of active compound perdose.

Methotrexate and other folate and antifolate agents have also beenemployed in the treastment of psoriasis, a disease characterized by anincreased rate epidermal cell proliferation. The compounds of formula 1,by virtue of functioning by the same underlying mechanisms are expectedto be valuable new agents in the treatment of psoriasis, theantineoplastic dosage is the same as the antipsoriasis dosage, exceptthat when the formula 1 compounds are used in the treatment of psoriasistopical application is preferred.

The compounds can be administered alone or in the form of apharmaceutical composition in combination with pharmaceuticallyacceptable carriers or excipients, the proportion and nature of whichare determined by the solubility and chemical properties of the compoundselected, the chosen route of administration, and standardpharmaceutical practice. The compounds of formula 1, while effectivethemselves, may be formulated and administered in the form of theirpharmaceutically acceptable acid addition salts for purposes ofstability, convenience of crystallization, increased solubility and thelike.

The compounds of formula 1 can be provided in compositions comprising anassayable amount of a compound of formula 1 in admixture with one ormore inert carriers. These compositions are useful, for example, asassay standards, as convenient means of making bulk shipments, or aspharmaceutical compositions. An assayable amount of a compound offormula 1 is an amount which is readily measurable by standard assayprocedures and techniques as are well known and appreciated by thoseskilled in the art. Assayable amounts of a compound of formula 1 willgenerally vary from about 0.001% to about 75% of the composition byweight. Inert carriers can be any material which does not degrade orotherwise covalently react with a compound of formula 1. Examples ofsuitable inert carriers are water; aqueous buffers, such as those whichare generally useful in High Performance Liquid Chromatography (HPLC)analysis; organic solvents, such as acetonitrile, ethyl acetate, hexaneand the like; and pharmaceutically acceptable carriers or excipients.These compositions are prepared by mixing the compound of formula 1 withthe inert carriers utilizing techniques and methods which are well knownand appreciated in the art.

More particularly, compounds of formula 1 can be provided inpharmaceutical compositions comprising a therapeutically effectiveantineoplastic amount of a compound of formula 1 in admixture orotherwise in association with one or more pharmaceutically acceptablecarriers or excipients.

The pharmaceutical compositions are prepared in a manner well known inthe pharmaceutical art. The carrier or excipient may be a solid,semi-solid, or liquid material which can serve as a vehicle or mediumfor the active ingredient. Suitable carriers or excipients are wellknown in the art. The pharmaceutical composition may be adapted for oralor parenteral use and may be administered to the patient in the form oftablets, capsules, suppositories, solution, suspensions, or the like.

The compounds of formula 1 may be administered orally, for example, withan inert diluent or with an edible carrier. They may be enclosed ingelatin capsules or compressed into tablets. For the purpose of oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of tablets, troches, capsules, elixirs,suspensions, syrups, wafers, chewing gums and the like. Thesepreparations should contain at least 4% of the compound of theinvention, the active ingredient, but may be varied depending upon theparticular form and may conveniently be between 4% to about 70% of theweight of the unit. The amount of the compound present in compositionsis such that a suitable dosage will be obtained. Preferred compositionsand preparations according to the present invention are prepared so thatan oral dosage unit form contains between 5.0-300 milligrams of acompound of the invention.

The tablets, pills, capsules, troches and the like may also contain oneor more of the following adjuvants: binders such as microcrystallinecellulose, gum tragacanth or gelatin; excipients such as starch orlactose, disintegrating agents such as alginic acid, Primogel, cornstarch and the like; lubricants such as magnesium stearate or Sterotex;glidants such as colloidal silicon dioxide; and sweetening agents suchas sucrose or saccharin may be added or a flavoring agent such aspeppermint, methyl salicylate or orange flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or a fatty oil. Otherdosage unit forms may contain other various materials which modify thephysical form of the dosage unit, for example, as coatings. Thus,tablets or pills may be coated with sugar, shellac, or other entericcoating agents. A syrup may contain, in addition to the presentcompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors. Materials used in preparing these variouscompositions should be pharmaceutically pure and nontoxic in the amountsused.

For the purpose of parenteral therapeutic administration, the compoundsof formula 1 may be incorporated into a solution or suspension. Thesepreparations should contain at least 0.1% of a compound of formula 1,but may be varied to be between 0.1 and about 50% of the weight thereof.The amount of the compound present in such compositions is such that asuitable dosage will be obtained. Preferred compositions andpreparations according to the present invention are prepared so that aparenteral dosage unit contains between 5.0 to 100 milligrams of thecompound of the invention.

The solutions or suspensions may also include the one or more of thefollowing adjuvants: sterile diluents such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylene diaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

As with any group of structurally related compounds which possesses aparticular generic utility, certain groups and configurations arepreferred for compounds of formula 1 in their end-use application.

Applicants prefer those compounds in which R₁ and R₂ are each a -NH₂group, X, Y and Z are each a nitrogen atom, and R₃ is a methyl group.Applicants also prefer those compounds in which R₁ is a --NH₂ group, R₂is an OH group, R₃ is a hydrogen, X and Z are each a -CH group and Y isa nitrogen atom.

EXAMPLE 1

N-[4-([(2,4-Diamino-6-pteridinyl)methyl]methylamino)-benzoyl]-4,4-D-Fluoroglutamicacid

A mixture of N-CH₃ PABA-F₂ Glu(OBu)₂ (0.201 mmol) and2,4-diaminopteridine-6-bromomethyl hydrobromide (75 mg, 0.223 mmol) inMe₂ NAc (2.5 ml) is stirred at 50-55° C. for 4 hr and then left atambient temperature overnight. Me₂ NAc is then removed under reducedpressure. This crude material is placed in an ice bath, and 2 ml oftrifluoroacetic acid is added with stirring. After 10 min, the reactionmixture is allowed to warm to ambient temperature and is stirred for 4hr. The trifluoroacetic acid is removed under reduced pressure givingthe crude product. The crude product is purified by chromatography onDEAE-cellulose with an NH₄ HCO₃ gradient (15-600 mM). Lyophilization ofthe column effluent containing the desired product gives the desiredpure product.

EXAMPLE 2

4-Amino-4-deoxy-10-methylpteroyl[D,L-erythro,threo-4,4-(difluoro)glutamic acid] (1, R₁, R₂═ NH_(2;) X,Y,Z═N;R₃═ CH₃)

In a 15 mL round bottom flask fitted with a drying tube is placed 2.5 mLDMF, 23 μL Et₃ N (0.16 mmol) and 25 μL (0.16 mmol) diethylphosphorocyanidate. The diaminopteroate (1,R₁, R₂═ NH_(2;) X,Y,Z═N;R₃═CH₃)(0.16 mmol) is then added and the reaction solution is stirred atambient temperature. After 3 h, an additional 5 μL (0.04 mmol) ofdiethyl phosphorocyanidate is added. F₂ Glu (0.16 mmol) is dissolved in1 mL DMF and added to the reaction flask and stirring continued for 72 hat ambient temperature. Solvent DMF is removed in vacuo, the residue isdissolved in 35 mL CHC13 and washed with 1% NH₄ OH (2×20 mL) to removeunreacted starting material. The organic layer is washed with 20 mL H₂O, then dried over Na₂ SO₄ and evaporated in vacuo to provide crudeblocked product. This coupled material is dissolved in neat TFA (3 mL)and progress of the de-esterification reaction is monitored by TLC.After 24 h, the solvent TFA is removed by rotary evaporation and theresidue dried in vacuo.

The crude product is dissolved in 20 mL H₂ O, the pH adjusted to 8 withdilute NH₄ OH, and the sample volume brought to 160 mL in order toobtain sufficiently low conductance prior to loading on a DEAE-cellulose(Whatman DE-52) column (30×1 cm). The column is washed with 100 mL waterand the desired formula 1 compound is eluted from the column with alinear gradient formed from 175 mL of 15 mM NH₄ HCO₃ and 175 mL of 500mM NH₄ HCO₃.

EXAMPLE 3

β,β-Difluoroglutamic acid bis-L-butyl ester 3a2,2-Difluoropent-4-enamide

To a stirred solution of 2,2-difluoropent-4-enoic acid (27.2 g, 0.2 mol)in hexane (200 mL) in a three-neck flask equipped with a refluxcondenser and N₂ inlet is added DMF (20 drops, catalytic) and oxalylchloride (20 mL, 0.23 mol). The mixture is stirred at room temperaturefor 2 h, when no further evolution of gas is seen. The solution iscooled in an ice/brine bath, the N₂ inlet is replaced by an empty CaCl₂tube (to trap any solid material blown out of the flask), and a streamof NH₃ gas is passed into the flask. The gas flow is maintained for 30min after the initial vigorous reaction has subsided. The mixture isthen poured into H₂ O (1 L) and Et₂ O (0.5 L). The glassware is washedwith H₂ O and Et₂ O, and the washings are added to this mixture. Celiteis added and insoluble material is removed by filtration. The phases areseparated. The Et₂ O phase is washed once with brine, dried over Na₂SO₄, and concentrated. The aqueous phase is extracted twice with CH₂Cl₂. The combined extracts are washed once with brine, dried over Na₂SO₄, combined with the residue of the ethereal phase, and concentrated.The residue is distilled to give the title amide (20.6 g, 76%) bp100-110° C./10 Torr, as a slightly yellow oil which solidifies oncooling, mp 33-35° C. ¹ H NMR (CDCl₃) δ 7.0-6.0 (2H, br.d), 5.75 (1 H,ddt, J=18, 9, 6 Hz), 5.3 (2 H, m), 2.87 (2 H, dt, J =6, 17 Hz). ¹⁹ F NMR(δ C₆ F₆₌ O) δ-56.0 (t, J =17 Hz).

Anal. Calcd for C₅ H₇ F₂ NO: C, 44.45; H, 5.22; N, 10.37. Found: C,42.03; H, 4.74; N, 9.33.

3b 2,2-Difluoropent-4-enonitrile

To a solution of the above amide (3a) (20.6 g, 0.152 mol) in drypyridine (25 mL, 0.31 mol) under N2 cooled in an ice/brine bath is addeddropwise over 11/2 h, trifluoroacetic anhydride (TFAA, 23.5 mL, 0.166mol). The reaction mixture becomes solid, and is allowed to stand atroom temperature for 4 h. The flask is then equipped for distillationunder N₂ and heated in an oil bath (finally at 130° C.). The distillateis collected to give 17.7 g of colorless oil which fumes in air, bp70-80° C./760 Torr. Analysis by NMR indicates that the composition ofthe distillate is nitrile:TFAA:pyridine 80:12:8. Hence the yield ofnitrile is 0.116 mol (76%). ¹ H NMR (CDCl₃) 6.0-5.5 (1 H, m), 5.4 (2 H,m), 2.93 (2 H, dt, J=6, 15 Hz). ¹⁹ F NMR (δ C₆ F₆₌ O) δ-71.7 (t, J =15Hz).

3c 4,4-Difluoroocta-1,6-dien-5-ylamine hydrochloride

Propenylmagnesium bromide is prepared by the addition of a solution of1-propenyl bromide (27.22 g, 0.225 mol) in THF (225 mL) to magnesiumturnings (5.47 g, 0.225 mmol) at such a rate as to maintain the mixturewarm but not boiling. When the addition is complete the mixture isstirred for 20 min, then diluted with THF (300 mL) and cooled to -15° C.To this mixture is added a solution of the nitrile (0.116 mmol) preparedin example 3b in THF (80 mL) at a rate such that the temperature of thereaction mixture remains within the range -15<T<-10° C. The mixture isstirred for a further 1 h in this temperature range, and then cooled to-40° C. A cold (-40° C.) solution of NaBH₄ (6.4 g, 0.169 mol) in MeOH(650 mL) and water (30 mL) is added in one portion. The cooling bath isremoved and the mixture is stirred for 2 h, then poured into 6 N HCl(800 mL). The organic solvents are removed by rotary eevaporation, andthe aqueous solution is washed twice with CH₂ Cl₂. The solution is thenbasified to pH 10 with NaOH pellets (ice-cooling is necessary), andsaturated with NaCl. Celite is added and the precipitated Mg(OH)₂ isremoved by filtration. The solid residue is washed with CH₂ Cl₂. Thefiltrate is extracted with CH₂ Cl₂ three times, and the combinedextracts and residue washings are dried over Na₂ SO₄, filtered,acidified with Et₂ O/HCl, and evaporated to give the title amine salt asa pale yellow solid (3 g). This material is not purified at this stage.

3d N-(t-Butyloxycarbonyl)-4,4-difluoroocta-1,6-dien-5-ylamine

The above crude salt of example 3c (35 g) is dissolved in water (200 mL)and dioxan (200 mL). Solid KHC03 is added to give a mixture which isneutral to pH paper, to which is added further KHC03 (12 g, 0.12 mol)and di-t-butyldicarbonate (34 g, 0.156 mol). The mixture is stirred atroom temperature overnight and then saturated with NaCl. The phases areseparated. The aqueous phase is extracted with hexane (×3). The extractsare combined with the organic phase and washed with water (×3). Thecombined aqueous washings are extracted once with hexane. This extractis combined with the organic phases which are then washed with brine,dried over Na₂ SO₄, and concentrated. The residue is purified by flashchromatography (eluant CH₂ Cl_(2/) pentane 1/1) to give the titlecarbamate (19 g, 65%), R_(f) 0.29, as an oil which soldifies onstanding. ¹ H NMR (CDCl₃) δ 6.0-5.5 (2 H, m); 5.5-4.5 (5 H, m); 2.67 (2H, dt, J=7, 16 Hz); 1.73 (3 H, dd*, J=7,1 Hz); 1.47 (9 H, s). ¹⁹ F NMR(δ C₆ F₆₌ 0) δ-53.2 (m).

3e β,β-Difluoroglutamic acid hydrochloride

To an ice-cold solution of KMnO₄ (52.56 g, 0.33 mol) in H₂ O (1.2 L) isadded a solution of the carbamate prepared in example 3d (10.95 g, 0.042mol) in AcOH (160 mL). The mixture is stirred overnight at roomtemperature, during which time a brown solid precipitates. Solid sodiumdisulphite is added to decolorize the mixture, followed by conc. HCl togive a solution of pH 2. This solution is extracted with Et₂ O (3×500mL), then saturated with NaCl and further extracted twice with Et₂ O.The combined organic fractions are washed once with brine andconcentrated. The residue is taken up in 1 N HCI (500 mL) andconcentrated. The residue is once again taken up in 1 N HCl, the mixtureis warmed, and activated charcoal is added. The mixture is stirred for10min, filtered, and the filtrate is evaporated to dryness. Extractionof the residue with hot isopropanol gives the crude amino acid (2.22 g).Further extraction of the isopropanol insoluble material with EtOH doesnot give any useful material. ¹⁹ F NMR (D₂ O: δ CF₃ CO₂ H=0) δ+23 (ddt,J=280, 5, 21 Hz),+27 (ddt, J=280, 22, 9 Hz); +28 (t); relative integrals5:5:3.

3f β,β-Difluoroglutamic acid bis-t-butyl ester

The crude acid of example 3e (2.22 g) is suspended in t-butyl acetate(500 mL), and HC104 (2.16 mL of 70% aqueous solution, 25 mmol) is addedto the mixture. The mixture is stirred at room temperature for 48 h, andthen extracted with H₂ O (2×200 mL). The combined extracts are washedwith CH₂ Cl₂ (2×100 mL) and then basified to pH 10 (4 N NaOH). Thesolution is extracted with CH₂ Cl₂ (3×100 mL). The combined extracts arewashed with brine, dried over Na₂ SO₄, and concentrated to one third oftheir volume. This solution can be used for the next step withoutfurther treatment. A sample evaporated more completely shows ¹ H NMR(CDCl₃) δ 4.10 (1 H, t, J =14 Hz), 3.10 (1 H, t, J =15 Hz), 3.00 (1 H,t, J= 15 Hz), 1.5 (18 H, 2 s); ¹⁹ F NMR (δ C₆ F₆₌ O) δ-58.7 (dt, J=14,15 Hz).

EXAMPLE 4

β,β-Difluoroqlutamic acid hydrochloride

To a solution of the BOC-bis-ester of example 3(300 mg, 0.76 mmol) indry Et₂ O (10 mL) is added saturated Et₂ O/HCl (3 mL). The mixture isstirred at room temperature (CaCl₂ tube protection) for 6 days. Theprecipitated white solid is collected and washed with Et₂ O. Analysis byTLC and NMR indicates that cleavage of the esters is not complete. Thematerial is taken up in 1 N HCl (5 mL) and stirred at room temperatureovernight. The reaction is still not complete. A few drops of conc. HClare added, and the mixture is stirred for 3 days. The water is removedin vacuo and the residue is dried by azeotropic evaporation with CCl₄.Trituration with diisopropyl ether (×2) gives a slightly green powder,which is collected and dried over P₂ O₅ to give the title amino acid(175 mg, 100%). ¹ H NMR (D₂ O). Signals obscured by solvent. ¹⁹ F NMR (δCF₃ CO₂ H═O) δ+23 (ddt, J =280, 5, 21 Hz), +27 (ddt, J =280, 22, 9 Hz).MS (CI, NH₃) m/e 184 (MH+, 100%), 140 (75%), 120 (40%), 102 (40%). Rf(HOAc/H20/BuOH 1/1/3) 0.19.

Anal. Calcd. for C₅ H₇ F₂ NO₄. HCl: C, 27.35: H, 3.67; N, 6.38. Found C,28.13; H, 3.58; N, 6.52.

We claim:
 1. A compound of the formula ##STR7## or a basic or an acidaddition salt thereof.
 2. A compound of the formula ##STR8## or a basicor an acid addition salt thereof.
 3. A compound of the formula ##STR9##wherein R₃ is H, (C₁₋ C₄)alkyl, allyl, or propargyl or a basic or anacid addition salt thereof.